Interaction of huntingtin fragments with brain membranes--clues to early dysfunction in Huntington's disease

Abstract Huntingtin is a large, multi-domain protein of unknown function in the brain. An abnormally elongated polyglutamine stretch in its N-terminus causes Huntington's disease (HD), a progressive neurodegenerative disorder. Huntingtin has been proposed to play a functional role in membrane trafficking via proteins involved in endo- and exocytosis. Here, we supply evidence for a direct association between huntingtin and membranes. In the brains of R6/2 mice with HD pathology, a 64 kDa N-terminal huntingtin fragment accumulated in postsynaptic membranes during the pre-symptomatic period of 4-8 weeks of age. In addition, an oligomeric fragment of approximately 200 kDa was detected at 8 weeks of age. Simultaneous progressive changes in distribution of amphiphysin, synaptojanin, and subunits of NMDA- and AMPA-receptors provide a strong indication of dysfunctional synaptic trafficking. Composition of the major phospholipids in the synaptic membranes was unaffected. In vitro, large unilamellar vesicles of brain lipids readily associated with soluble N-terminal huntingtin exon 1 fragments and stimulated fibrillogenesis of mutant huntingtin aggregates. Moreover, interaction of both mutant and wild-type huntingtin exon 1 fragments with brain lipids caused bilayer perturbation, mediated through a proline-rich region adjacent to the polyglutamines. This suggests that lipid interactions in vivo could influence misfolding of huntingtin and may play an early role in HD pathogenesis.     

Huntingtin is required for ER-to-Golgi transport and for secretory vesicle fusion at the plasma membrane

Huntingtin is a large membrane-associated scaffolding protein that associates with endocytic and exocytic vesicles and modulates their trafficking along cytoskeletal tracks. Although the progression of Huntington’s disease is linked to toxic accumulation of mutant huntingtin protein, loss of wild-type huntingtin function might also contribute to neuronal cell death, but its precise function is not well understood. Therefore, we investigated the molecular role of huntingtin in exocytosis and observed that huntingtin knockdown in HeLa cells causes a delay in endoplasmic reticulum (ER)-to-Golgi transport and a reduction in the number of cargo vesicles leaving the trans-Golgi network. In addition, we found that huntingtin is required for secretory vesicle fusion at the plasma membrane. Similar defects in the early exocytic pathway were observed in primary fibroblasts from homozygous Htt^140Q/140Q knock-in mice, which have the expansion inserted into the mouse huntingtin gene so lack wild-type huntingtin expression. Interestingly, heterozygous fibroblasts from a Huntington’s disease patient with a 180Q expansion displayed no obvious defects in the early secretory pathway. Thus, our results highlight the requirement for wild-type huntingtin at distinct steps along the secretory pathway.KEY WORDS: Exocytosis, Huntingtin, ER, Golgi, Vesicle fusion     

A stress sensitive ER membrane-association domain in Huntingtin protein defines a potential role for Huntingtin in the regulation of autophagy

We have recently published the precise definition of an aminoterminal membrane association domain in huntingtin, capable of targeting to the endoplasmic reticulum and late endosomes as well as autophagic vesicles. In response to ER stress induced by several pathways, huntingtin releases from membranes and rapidly translocates into the nucleus. Huntingtin is then capable of nuclear export and re-association with the ER in the absence of stress. This release is inhibited when huntingtin contains the polyglutamine expansion seen in Huntington's disease. As a result, mutant huntingtin expressing cells have a perturbed ER and an increase in autophagic vesicles. Here, we discuss the potential function of the huntingtin protein as an ER sentinel, potentially regulating autophagy in response to ER stress. We compare these recent findings to the well characterized mammalian target of rapamycin, mTor, a protein described over a decade ago as related to huntingtin structurally by leucine-rich, repetitive HEAT sequences. Since then, the described functional similarities between Huntingtin and mTor are striking, and this new information about huntingtin's direct association with autophagic vesicles indicates that this structural similarity may extend to functional similarities having an impact upon ER functionality and autophagy.     

A specific product of phosphatidylinositol 3-kinase directly activates the protein kinase Akt through its pleckstrin homology domain.

Phosphatidylinositol (PI) 3-kinase is a cytoplasmic signaling molecule that is recruited to activated growth factor receptors after growth factor stimulation of cells. Activation of PI 3-kinase results in increased intracellular levels of 3' phosphorylated inositol phospholipids and the induction of signaling responses, including the activation of the protein kinase Akt, which is also known as RAC-PK or PKB. We tested the possibility that the phospholipid products of PI 3-kinase directly mediate the activation of Akt. We have previously described a constitutively active PI 3-kinase, p110, which can stimulate Akt activity. We used purified p110 protein to generate a series of 3' phosphorylated inositol phospholipids and tested whether any of these lipids could activate Akt in vitro. Phospholipid vesicles containing PI3,4 bisphosphate (P2) specifically activated Akt in vitro. By contrast, the presence of phospholipid vesicles containing PI3P or PI3,4,5P3 failed to increase the kinase activity of Akt. Akt could also be activated by synthetic dipalmitoylated PI3,4P2 or after enzymatic conversion of PI3,4,5P3 into PI3,4P2 with the signaling inositol polyphosphate 5' phosphatase SIP. We show that PI3,4P2-mediated activation is dependent on a functional pleckstrin homology domain in Akt, since a point mutation in the pleckstrin homology domain abrogated the response to PI3,4P2. Our findings show that a phospholipid product of PI 3-kinase can directly stimulate an enzyme known to be an important mediator of PI 3-kinase signaling.     

Direct activation of the epithelial Na(+) channel by phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate produced by phosphoinositide 3-OH kinase

The phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) is accepted to be a direct modulator of ion channel activity. The products of phosphoinositide 3-OH kinase (PI3K), PtdIns(3,4)P(2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), in contrast, are not. We report here activation of the epithelial Na(+) channel (ENaC) reconstituted in Chinese hamster ovary cells by PI3K. Insulin-like growth factor-I also activated reconstituted ENaC and increased Na(+) reabsorption across renal A6 epithelial cell monolayers via PI3K. Neither IGF-I nor PI3K affected the levels of ENaC in the plasma membrane. The effects of PI3K and IGF-I on ENaC activity paralleled changes in the plasma membrane levels of the PI3K product phospholipids, PtdIns(3,4)P(2)/PtdIns(3,4,5)P(3), as measured by evanescent field fluorescence microscopy. Both PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) activated ENaC in excised patches. Activation of ENaC by PI3K and its phospholipid products corresponded to changes in channel open probability. We conclude that PI3K directly modulates ENaC activity via PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3). This represents a novel transduction pathway whereby growth factors, such as IGF-I, rapidly modulate target proteins independent of signaling elicited by kinases downstream of PI3K.     

Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.     

Calcineurin is involved in the early activation of NMDA-mediated cell death in mutant huntingtin knock-in striatal cells

Excitotoxicity has been proposed as one of the mechanisms involved in the specific loss of striatal neurons that occurs in Huntington's disease. Here, we studied the role of calcineurin in the vulnerability of striatal neurons expressing mutant huntingtin to excitotoxicity. To this end, we induced excitotoxicity by adding NMDA to a striatal precursor cell line expressing full-length wild-type (STHdh^Q7/Q7) or mutant (STHdh^Q111/Q111) huntingtin. We observed that cell death appeared earlier in STHdh^Q111/Q111 cells than in STHdh^Q7/Q7 cells. Interestingly, these former cells expressed higher levels of calcineurin A that resulted in a greater increase of its activity after NMDA receptor stimulation. Moreover, transfection of full-length mutant huntingtin in different striatal-derived cells (STHdh^Q7/Q7, M213 and primary cultures) increased calcineurin A protein levels. To determine whether high levels of calcineurin A might account for the earlier activation of cell death in mutant huntingtin knock-in cells, wild-type cells were transfected with calcineurin A. Calcineurin A-transfected STHdh^Q7/Q7 cells displayed a significant increase in cell death compared with that recorded in green fluorescent protein-transfected cells after NMDA treatment. Notably, addition of the calcineurin inhibitor FK-506 produced a more robust reduction in cell death in mutant huntingtin knock-in cells than it did in wild-type cells. These results suggest that high levels of calcineurin A could account for the increased vulnerability of striatal cells expressing mutant huntingtin to excitotoxicity.     

NOREPINEPHRINE INCREASES THE [32P]LABELLING OF A SPECIFIC PHOSPHOLIPID FRACTION OF POST-SYNAPTIC PINEAL MEMBRANES

Abstract— Cultured pineal glands incorporated ³²P into membrane phospholipids. Treatment of cultured glands with norepinephrine, which is known to stimulate membrane- bound pineal adenyl cyclase and to increase the production and secretion of melatonin, stimulated the incorporation of ³²P into a phospholipid fraction of membranes and particulates containing phosphatidyl serine and phosphatidyl inositol. The labelling of other phospholipid fractions and the total ³²P in the gland were not changed by norepinephrine treatment. Experiments with chronically-denervated pineal glands indicated that the effect of norepinephrine on the [³²P]labelling of phospholipids occurred at a postsynaptic site. When norepinephrine-stimulated secretion of melatonin was partially inhibited by p -chlorophenylalanine (a compound which blocks the synthesis of melatonin precursors), the norepinephrine-stimulated labelling of phospholipids was still observed. Conversely, when melatonin secretion was stimulated in the absence of norepinephrine by treatment with the immediate precursor of melatonin, N -acetylserotonin, a stimulation of ³²P- labelling of phospholipids did not occur. These observations suggest that the increased [³²P]- labelling of a phospholipid fraction caused by the norepinephrine treatment is not related to the secretion of melatonin. This effect on phospholipids may be associated with the interaction of norepinephrine with a membrane-bound postsynaptic receptor. Stimulation by norepinephrine of [³²P]-incorporation into phospholipids has not been previously reported to occur in a tissue in which cholinergic fibres are absent.     

Huntingtin aggregation kinetics and their pathological role in a Drosophila Huntington's disease model

Huntington's disease is a neurodegenerative disorder resulting from expansion of a polyglutamine tract in the Huntingtin protein. Mutant Huntingtin forms intracellular aggregates within neurons, although it is unclear whether aggregates or more soluble forms of the protein represent the pathogenic species. To examine the link between aggregation and neurodegeneration, we generated Drosophila melanogaster transgenic strains expressing fluorescently tagged human huntingtin encoding pathogenic (Q138) or nonpathogenic (Q15) proteins, allowing in vivo imaging of Huntingtin expression and aggregation in live animals. Neuronal expression of pathogenic Huntingtin leads to pharate adult lethality, accompanied by formation of large aggregates within the cytoplasm of neuronal cell bodies and neurites. Live imaging and Fluorescence Recovery After Photobleaching (FRAP) analysis of pathogenic Huntingtin demonstrated that new aggregates can form in neurons within 12 hr, while preexisting aggregates rapidly accumulate new Huntingtin protein within minutes. To examine the role of aggregates in pathology, we conducted haplo-insufficiency suppressor screens for Huntingtin-Q138 aggregation or Huntingtin-Q138-induced lethality, using deficiencies covering ~80% of the Drosophila genome. We identified two classes of interacting suppressors in our screen: those that rescue viability while decreasing Huntingtin expression and aggregation and those that rescue viability without disrupting Huntingtin aggregation. The most robust suppressors reduced both soluble and aggregated Huntingtin levels, suggesting toxicity is likely to be associated with both forms of the mutant protein in Huntington's disease.     

Phosphoinositide 3-kinase: A new effector in signal transduction?

Interest in phosphopinositide 3-kinase (PI 3-kinase) has been fuelled by its identification as a major phosphotyrosyl protein detected in cells following growth factor stimulation and oncogenic transformation. It is found complexed with activated growth factor receptors and non-receptor tyrosine kinases, thus suggesting that it participates in the signal transduction pathways initiated by the activation of tyrosine kinases. PI 3-kinase phosphorylates the 3-position in the inositol ring of the well known inositol phospholipids in vitro giving phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate [PtdIns3P, PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃], respectively. The cellular levels of PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ rapidly increase in circumstances where PI 3-kinase becomes complexed with tyrosine kinases. Accumulation of the same lipids also occurs in platelets and neutrophils following stimulation of G-protein linked -thrombin and chemotactic peptide receptors, respectively, leading to speculation that one or both of these lipids is a new second messenger whose function is not yet known. This review brings together recent information on the isolation, characterization and regulation of PI 3-kinase, the cellular occurrence of 3-phosphorylated inositol phospholipids and possible functions of the PI 3-kinase pathway in cell signalling.     

Specific membrane binding of the carboxypeptidase Y inhibitor I(C), a phosphatidylethanolamine-binding protein family member

I(C), an endogenous cytoplasmic inhibitor of vacuolar carboxypeptidase Y in the yeast Saccharomyces cerevisiae, is classified as a member of the phosphatidylethanolamine-binding protein family. The binding of I(C) to phospholipid membranes was first analyzed using a liposome-binding assay and by surface plasmon resonance measurements, which revealed that the affinity of this inhibitor was not for phosphatidylethanolamine but for anionic phospholipids, such as phosphatidylserine, phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate, with K(D) values below 100 nm. The liposome-binding assay and surface plasmon resonance analyses of I(C), when complexed with carboxypeptidase Y, and the mutant forms of I(C) further suggest that the N-terminal segment (Met1-His18) in its carboxypeptidase Y-binding sites is involved in the specific and efficient binding to anionic phospholipid membranes. The binding of I(C) to cellular membranes was subsequently analyzed by fluorescence microscopy of yeast cells producing the green fluorescent protein-tagged I(C), suggesting that I(C) is specifically targeted to vacuolar membranes rather than cytoplasmic membranes, during the stationary growth phase. The present findings provide novel insights into the membrane-targeting and biological functions of I(C) and phosphatidylethanolamine-binding proteins.     

Huntingtin phosphorylation acts as a molecular switch for anterograde/retrograde transport in neurons

The transport of vesicles in neurons is a highly regulated process, with vesicles moving either anterogradely or retrogradely depending on the nature of the molecular motors, kinesins and dynein, respectively, which propel vesicles along microtubules (MTs). However, the mechanisms that determine the directionality of transport remain unclear. Huntingtin, the protein mutated in Huntington's disease, is a positive regulatory factor for vesicular transport. Huntingtin is phosphorylated at serine 421 by the kinase Akt but the role of this modification is unknown. Here, we demonstrate that phosphorylation of wild-type huntingtin at S421 is crucial to control the direction of vesicles in neurons. When phosphorylated, huntingtin recruits kinesin-1 to the dynactin complex on vesicles and MTs. Using brain-derived neurotrophic factor as a marker of vesicular transport, we demonstrate that huntingtin phosphorylation promotes anterograde transport. Conversely, when huntingtin is not phosphorylated, kinesin-1 detaches and vesicles are more likely to undergo retrograde transport. This also applies to other vesicles suggesting an essential role for huntingtin in the control of vesicular directionality in neurons.     

Enhancement of phototransduction g protein-effector interactions by phosphoinositides

Light responses in photoreceptor cells are mediated by the action of the G protein transducin (G(t)) on the effector enzyme cGMP phosphodiesterase (PDE6) at the surface of disk membranes. The enzymatic components needed for phosphoinositide-based signaling are known to be present in rod cells, but it has remained uncertain what role phosphoinositides play in vertebrate phototransduction. Reconstitution of PDE6 and activated G(alphat), on the surface of large unilamellar vesicles containing d-myo-phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)), stimulated PDE activity nearly 4-fold above the level observed with membranes containing no phosphoinositides, whereas G protein-independent activation by trypsin was unaffected by the presence of phosphoinositides. PDE activity was similarly stimulated by d-myo-phosphatidylinositol-3,4-bisphosphate and d-myo-phosphatidylinositol-4-phosphate (PI(4)P), but much less by d-myo-phosphatidylinositol-5-phosphate (PI(5)P) or d-myo-phosphatidylinositol-3,5-bisphosphate. Incubation of rod outer segment membranes with phosphoinositide-specific phospholipase C decreased G protein-stimulated activation of endogenous PDE6, but not trypsin-stimulated PDE activity. Binding experiments using phosphoinositide-containing vesicles revealed patterns of PDE6 binding and PDE6-enhanced G(alphat)-GTPgammaS binding, consistent with the activation profile PI(4,5)P(2) > PI(4)P > PI(5)P approximately control vesicles. These results suggest that enhancement of effector-G protein interactions represents a possible mechanism for modulation of phototransduction gain by changes in phosphoinositide levels, perhaps occurring in response to longterm changes in illumination or other environmental cues.     

Anionic phospholipids are involved in membrane targeting of PI 3-kinase gamma

Phosphoinositide 3-kinases (PI 3-kinases) have critical roles in diverse cellular signaling processes and in protein trafficking. In contrast to the class I PI 3-kinases alpha, beta, and delta which bind via src homology 2 (SH2) domains of adaptor proteins to tyrosine kinase receptors, the mechanism of recruitment of the PI 3-kinase gamma to membranes is unknown. We report in vitro experiments using immobilized proteins and small unilamellar vesicles which suggest an involvement of anionic phospholipids in membrane association of PI 3-kinase gamma. Furthermore we provide evidence that the enzyme displays beside the catalytic center a phospholipid binding domain which is essential for enzyme activity.     

Disease-toxicant screen reveals a neuroprotective interaction between Huntington's disease and manganese exposure

Recognizing the similarities between Huntington's disease (HD) pathophysiology and the neurotoxicology of various metals, we hypothesized that they may exhibit disease-toxicant interactions revealing cellular pathways underlying neurodegeneration. Here, we utilize metals and the ST Hdh mouse striatal cell line model of HD to perform a gene–environment interaction screen. We report that striatal cells expressing mutant Huntingtin exhibit elevated sensitivity to cadmium toxicity and resistance to manganese toxicity. This neuroprotective gene–environment interaction with manganese is highly specific, as it does not occur with iron, copper, zinc, cobalt, cadmium, lead, or nickel ions. Analysis of the Akt cell stress signaling pathway showed diminished activation with manganese exposure and elevated activation after cadmium exposure in the mutant cells. Direct examination of intracellular manganese levels found that mutant cells have a significant impairment in manganese accumulation. Furthermore, YAC128Q mice, a HD model, showed decreased total striatal manganese levels following manganese exposure relative to wild-type mice. Thus, this disease-toxicant interaction screen has revealed that expression of mutant Huntingtin results in heightened sensitivity to cadmium neurotoxicity and a selective impairment of manganese accumulation.     

Aggregate-centered redistribution of proteins by mutant huntingtin

Huntingtin is a widely expressed 350-kDa cytosolic multidomain of unknown function. Aberrant expansion of the polyglutamine tract located in the N-terminal region of huntingtin results in Huntington's disease. The presence of insoluble huntingtin inclusions in the brains of patients is one of the hallmarks of Huntington's disease. Experimentally, both full-length huntingtin and N-terminal fragments of huntingtin with expanded polyglutamine tracts trigger aggregate formation. Here, we report that upon the formation of huntingtin aggregates; endogenous cytosolic huntingtin, Hsc70/Hsp70 (heat shock protein and cognate protein of 70kDa) and syntaxin 1A become aggregate-centered. This redistribution suggests that these proteins are eventually depleted and become unavailable for normal cellular function. These results indicate that the cellular targeting of several key proteins are altered in the presence of mutant huntingtin and suggest that aggregate depletion of these proteins may underlie, in part, the sequence of disease progression.     

Polyglutamine expansion in huntingtin increases its insertion into lipid bilayers

An expanded polyglutamine (Q) tract (>37Q) in huntingtin (htt) causes Huntington disease. Htt associates with membranes and polyglutamine expansion in htt may alter membrane function in Huntington disease through a mechanism that is not known. Here we used differential scanning calorimetry to examine the effects of polyQ expansion in htt on its insertion into lipid bilayers. We prepared synthetic lipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine and tested interactions of htt amino acids 1-89 with 20Q, 32Q or 53Q with the vesicles. GST-htt1-89 with 53Q inserted into synthetic lipid vesicles significantly more than GST-htt1-89 with 20Q or 32Q. We speculate that by inserting more into cell membranes, mutant huntingtin could increase disorder within the lipid bilayer and thereby disturb cellular membrane function.     

Liposomes can function as targets for natural killer cytotoxic factor but not for tumor necrosis factor

NK cells exert their lytic action through the release of NK cytotoxic factors (NKCF) after stimulation by the bound target cell. NKCF may be related to granule-derived perforin/cytolysin on one hand and to the pleiotropic cytokine TNF on the other hand. In the present study, we show that NKCF can also lyse artificial lipid vesicles, as had been reported previously for cytotoxic granules and cytolysin. The lysis of large unilamellar vesicles was monitored by measuring the release of the encapsulated fluorescent dye carboxyfluorescein. NKCF-induced lysis was only observed with liposomes composed of a complex mixture of lipids including acidic phospholipids. No lysis could be demonstrated if the liposomes contained phosphatidylcholine as the only phospholipid, suggesting some kind of lipid specificity for the action of NKCF. A remarkable finding was that neither recombinant nor natural TNF were able to lyse large unilamellar vesicles, irrespective of their lipid composition, indicating different ways of interaction of NKCF and TNF with artificial (and presumably also biological) membranes.